If you only want to do this once for presentation purpose, you can use the function "Viewer" during test mode. Then save it manually, shown below:Note: you may encounter some freeze in Viewer, simply restart CellProfiler. So save your pipeline first before using "Viewer".

If you would like to repeat this overlay for every images of the pipeline, the best practice would be making 2 separate pipelines:

First pipeline contains the following modules: IndentifyObjects > "SaveImages" for red objects, and another "SaveImages" for green objects.You can continue to do all needed measurements here.

In second pipeline, when you load the images, you would load both the brightfield image and the two newly saved red, green images. Note: set them as "color" images in module "NamesAndTypes". Then use "ImageMath", add brightfield+green+red together > use module "SaveImages" save the ImageAfterMath, which now contain all three colors

Thank you. I am going to give this a shot. Somewhat disappointed i will have to use two pipelines. Although, my current pipeline is getting pretty long so this may be an opportunity to spilt it up between image creation and image quantification.

I don't think you should NEED two pipelines- you could probably in your original pipeline use GrayToColor on the red+green channels to make them into one RGB image (and just leave the blue as blank), then with ImageMath add it to the brightfield. I haven't tested this though, so take with a grain of salt.

My Pipeline is a mess, and i dont have the time to clean it up for this post. So if in the future someone need help with this know that it can be done. And comment on this post for my help.

My BF was a single channel, so i used GrayToColor-> each channel was red:BF; green:BF, blue:BFRescaleIntensity -> BF_2, stretch image to full intensity rangeGrayToColor-> Red:Tritc; Green;GFP; Blue:blankRescaleIntensity -> tritc_gfp_rs, stretch image to full intensity rangeImageMath-> BF_rs + tritc_gfp_rs, ALL DEFAULT VALUES EXCEPT set values greater that 1 to 1. If this is not done you will have a blue background